Protein kinase C (PKC) is a key enzyme in signal transduction involved in a variety of cellular functions, including cell growth, regulation of gene expression and ion channel activity. The PKC family of isozymes includes at least 11 different protein kinases which can be divided into at least three subfamilies based on their homology and sensitivity to activators. Members of the classical or cPKC subfamily, α, βI, βII and γPKC, contain four homologous domains (C1, C2, C3 and C4) inter-spaced with isozyme-unique (variable or V) regions, and require calcium, phosphatidylserine (PS), and diacylglycerol (DG) or phorbol esters for activation. Members of the novel or nPKC subfamily, δ, ε, η, and θPKC, lack the C2 homologous domain and do not require calcium for activation. Finally, members of the atypical or αPKC subfamily, ζ and λ/ι PKC, lack both the C2 and one half of the C1 homologous domains and are insensitive to DG, phorbol esters and calcium.
Studies on the subcellular distribution of PKC isozymes demonstrate that activation of PKC results in its redistribution in the cells (also termed translocation), such that activated PKC isozymes associate with the plasma membrane, cytoskeletal elements, nuclei, and other subcellular compartments (Saito, N., et al., Proc. Natl. Acad. Sci. USA 86:3409-3413 (1989); Papadopoulos, V. and Hall, P. F., J. Cell Biol. 108:553-567 (1989); Mochly-Rosen, D., et al., Molec. Biol. Cell (formerly Cell Reg.) 1:693-706 (1990)).
It appears that the unique cellular functions of different PKC isozymes are determined by their subcellular location. For example, activated βIPKC is found inside the nucleus, whereas activated βIIPKC is found at the perinucleus and cell periphery of cardiac myocytes (Disatnik, M. H., et al., Exp. Cell Res. 210:287-297 (1994)). Further, in the same cells, εPKC binds to cross-striated structures (possibly the contractile elements) and cell-cell contacts following activation or after addition of exogenous activated εPKC to fixed cells (Mochly-Rosen, et al., 1990; Disatnik, et al., 1994). The localization of different PKC isozymes to different areas of the cell in turn appears due to binding of the activated isozymes to specific anchoring molecules termed Receptors for Activated C-Kinase (RACKs).
RACKs are thought to function by selectively anchoring activated PKC isozymes to their respective subcellular sites. RACKs bind only fully activated PKC and are not necessarily substrates of the enzyme. Nor is the binding to RACKs mediated via the catalytic domain of the kinase (Mochly-Rosen, D., et al., Proc. Natl. Acad. Sci. USA 88:3997-4000 (1991)). Translocation of PKC reflects binding of the activated enzyme to RACKs anchored to the cell particulate fraction and the binding to RACKs is required for PKC to produce its cellular responses (Mochly-Rosen, D., et al., Science 268:247-251 (1995)). Inhibition of PKC binding to RACKs in vivo inhibits PKC translocation and PKC-mediated function (Johnson, J. A., et al., J. Biol. Chem 271:24962-24966 (1996a), Ron, D., et al., Proc. Natl. Acad. Sci. USA 92:492-496 (1995); Smith, B. L. and Mochly-Rosen, D., Biochem. Biophys. Res. Commun. 188:1235-1240 (1992)).
cDNA clones encoding RACK1 and RACK2 have been identified (U.S. Pat. No. 5,519,003; Ron, D., et al., Proc. Natl. Acad. Sci. USA 91:839-843 (1994); Csukai, M., et al., 9TH INTERNATIONAL CONFERENCE ON SECOND MESSENGERS AND PHOSPHOPROTEINS 112 (1995)). Both are homologs of the β subunit of G proteins, a receptor for another translocating protein kinase, the β-adrenergic receptor kinase, βARK (Pitcher, J., et al., Science 257:1264-1267 (1992)). Similar to Gβ, RACK1, and RACK2 have seven WD40 repeats (Ron, et al., 1994; Csukai, et al., 1995). Recent data suggest that RACK1 is a βIIPKC-specific RACK (Stebbins, E. G., et al., J. Biol. Chem. 276:29644-29650 (2001)) and that RACK2 (Csukai, M., et al, J. Biol. Chem. 272:29200-29206 (1997)) is specific for activated εPKC.
Translocation of PKC is required for proper function of PKC isozymes. Peptides that mimic either the PKC-binding site on RACKs (Mochly-Rosen, D., et al., J. Biol. Chem., 226:1466-1468 (1991a); Mochly-Rosen, et al., 1995) or the RACK-binding site on PKC (Ron, et al., 1995; Johnson, et al., 1996a) are isozyme-specific translocation inhibitors of PKC that selectively inhibit the function of the enzyme in vivo. For example, an eight amino acid peptide derived from εPKC (peptide εV1-2; SEQ ID NO:1, Glu Ala Val Ser Leu Lys Pro Thr) is described in U.S. Pat. No. 6,165,977. The peptide contains a part of the RACK-binding site on εPKC and selectively inhibits specific εPKC-mediated functions in cardiac myocytes. This εPCK peptide has been shown to be involved in cardiac preconditioning to provide protection from ischemic injury. Prolonged ischemia causes irreversible myocardium damage primarily due to death of cells at the infarct site. Studies in animal models, isolated heart preparations and isolated cardiac myocytes in culture have demonstrated that short bouts of ischemia of cardiac muscle reduce such tissue damage in subsequent prolonged ischemia (Liu, Y., et al., J. Mol. Cell. Cardiol. 27:883-892 (1995); Hu, K. and Nattel, S., Circulation 92:2259-2265 (1995); Brew, E. C., et al., Am. J. Physiol 269(Heart Circ. Physiol. 38):H1370-H1378 (1995); Schultz, J. E. J., et al., Circ. Res. 78:1100-1104 (1996)). This protection, which occurs naturally following angina and has been termed preconditioning, can be mimicked by a variety of non-specific PKC agonists (Mitchell, M. B., et al., Circulation 88:1633 (1993); Mitchell, M. B., et al., Circ. Res. 76:73-81 (1995); Murry, C. E., et al., Circulation 74:1123-1136 (1986); Speechly-Dick, M. E., et al., Circ. Res. 75:586-590 (1993)). Both δPKC and εPKC activation occurs following preconditioning (Gray, M. O. et al., J. Biol. Chem. 272:30945-3095 (1997)), however, εPKC activation is required for protection of cardiac myocytes from ischemia-induced cell death (U.S. Pat. No. 6,165,977).
In a recent study, an εPKC-selective peptide agonist was shown to provide cardio-protection from ischemia when administered intracellulary to isolated neonatal and adult cardiomyocytes and when produced intracellulary in vivo in transgenic mice (Dorn, G., Proc. Natl. Acad. Sci. USA 96(22):12798-12803 (1999)).
The ability of δPCK peptide agonists and antagonists to protect cells and tissue from an ischemic event or to reverse or reduce damage caused by an ischemic event has not been reported. More particularly, it is unknown in the art whether or not δPCK peptide agonists and antagonists can be delivered extracellulary to whole tissue or intact organs in vivo to achieve a therapeutic effect.